DOCSLIB.ORG

Molecular Genetic and Physical Analysis of Gas Vesicles in Buoyant Enterobacteria

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Molecular Genetic and Physical Analysis of Gas Vesicles in Buoyant Enterobacteria View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Apollo Environmental Microbiology (2016) 18(4), 1264–1276 doi:10.1111/1462-2920.13203 doi:10.1111/1462-2920.13203 Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria Yosuke Tashiro,1,2† Rita E. Monson,1† bacterial cells; the first time this has been performed Joshua P. Ramsay1,3 and George P. C. Salmond1* in either strain. 1Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK. Introduction 2 Applied Chemistry and Biochemical Engineering Bacteria can use multiple modes of taxis, including swim- Course, Department of Engineering, Graduate School of ming, twitching, swarming and flotation. Flotation is Integrated Science and Technology, Shizuoka enabled through the regulated production of intracellular University, Hamamatsu 432-8561, Japan. buoyancy chambers: gas vesicles. Gas vesicle-driven 3 Curtin Health Innovation Research Institute Biosciences buoyancy facilitates the movement of photosynthetic Precinct, Faculty of Health Sciences, Curtin University, cyanobacteria into water column positions, allowing expo- Bentley, WA 6102, Australia. sure to wavelengths of light that can support phototrophy (Pfeifer, 2012). Gas vesicles can also increase bacterial Summary surface area-to-volume ratios, and thereby help survival under environmental and nutritional stresses (Houwink, Different modes of bacterial taxis play important roles 1956; Walsby, 1972). in environmental adaptation, survival, colonization Gas vesicles are visible as light-refracting structures and dissemination of disease. One mode of taxis is under phase-contrast microscopy (PCM), and bacterial flotation due to the production of gas vesicles. Gas colonies producing gas vesicles can be opaque on plates. vesicles are proteinaceous intracellular organelles, All gas vesicles identified to date appear to be constructed permeable only to gas, that enable flotation in aquatic from homologous proteins (Pfeifer, 2012). The primary niches. Gene clusters for gas vesicle biosynthesis gas vesicle structural protein, GvpA, assembles into are partially conserved in various archaea, tandem arrays that form ribs of the cylindrical vesicle cyanobacteria, and some proteobacteria, such as the (Englert and Pfeifer, 1993; Walsby, 1994). A second enterobacterium, Serratia sp. ATCC 39006 (S39006). protein, GvpC, forms an exterior mesh on the gas vesicle Here we present the first systematic analysis of the surface, providing further structural support and influenc- genes required to produce gas vesicles in S39006, ing gas vesicle shape (Hayes et al., 1988; 1992; Walsby identifying how this differs from the archaeon and Hayes, 1988; Englert and Pfeifer, 1993; Walsby, Halobacterium salinarum. We define 11 proteins 1994; Offner et al., 2000). Recently, the structure of GvpF essential for gas vesicle production. Mutation of gvpN from the cyanobacterium, Microcystis aeruginosa was or gvpV produced small bicone gas vesicles, sug- solved, and through electron microscopy, GvpF was gesting that the cognate proteins are involved in the shown to form a part of the gas vesicle structure (Xu et al., morphogenetic assembly pathway from bicones to 2014). However, the precise biochemical roles of many mature cylindrical forms. Using volumetric compres- other proteins found in gas vesicle gene clusters are sion, gas vesicles were shown to comprise 17% unknown, although their stoichiometry is thought to be of S39006 cells, whereas in Escherichia coli important (Shukla and DasSarma, 2004; Chu et al., 2011; heterologously expressing the gas vesicle cluster in a Pfeifer, 2012). deregulated environment, gas vesicles can occupy Recently, we discovered gas vesicles in the enterobac- around half of cellular volume. Gas vesicle produc- terium, Serratia spp. ATCC39006 (S39006), a genetically tion in S39006 and E. coli was exploited to calculate tractable host (Ramsay et al., 2011). S39006 is a the instantaneous turgor pressure within cultured Gram-negative bacillus that is pathogenic to plant and nematode hosts and produces two secondary metabolites: the tripyrrole red pigment, 2-methyl-3- pentyl-6-methoxyprodigiosin (prodigiosin) and the Received 9 November, 2015; accepted 29 December, 2015. *For β correspondence. E-mail [email protected]; Tel. +01223 333650; Fax -lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (a +01223 766108. †These authors contributed equally to this work. carbapenem) (Coulthurst et al., 2005; Williamson et al., V©C 2016 The Authors. Environmental Microbiology published published by by Society Society for for Applied Applied Microbiology Microbiology and and John John Wiley Wiley & & Sons Sons Ltd. Ltd. This is an open access article under the terms of the Creative Commons Attribution License, License, which which permits permits use, use, distribution distribution and and reproduction in any medium, provided provided the the original original work work is is properly properly cited. cited. 2 Y. Tashiro, R. E. Monson, J. P. Ramsay and G. P. C.Characterization Salmond of gas vesicles in enterobacteria 1265 2006). The genetic locus for gas vesicle biosynthesis in GvpA domain. In addition, S39006 has three genes S39006 was determined, and gas vesicles were shown to (gvpF1, gvpF2 and gvpF3) encoding homologues of a be responsible for the opaque colony phenotype and reportedly minor structural protein, GvpF, and each is buoyancy. Transcription of the gas vesicle gene cluster is conserved in γ- and δ-proteobacteria that carry a gas cell density-dependent [under the control of the SmaIR vesicle gene cluster (Fig. S1). Other genes encoding the quorum-sensing (QS) system], responsive to oxygen gas vesicle-associated proteins GvpC, GvpG, GvpH, status, and controlled by the gas vesicle regulatory GvpK and GvpN are also conserved in seven protein A, GvrA (a member of the NtrC family of regula- proteobacteria (Fig. 1 and Fig. S1). S39006 has five can- tors) encoded within the gas vesicle cluster (Ramsay didate gas vesicle proteins of unknown function: GvpV, et al., 2011; Ramsay and Salmond, 2012). Although there GvpW, GvpX, GvpY and GvpZ, and of these, GvpV and are obvious similarities between various gas vesicle gene GvpZ are conserved in four other β-, γ- and clusters, the S39006 cluster does not encode the two δ-proteobacteria gas vesicle gene clusters (Fig. 1). widely studied regulatory proteins, GvpD and GvpE, that are found in Halobacterium salinarum strains and in The gas vesicle locus in S39006 is defined by two Haloferax mediterranei (Englert et al., 1992; Ng et al., distinct operons; one directly regulated by the QS 2000; Zimmermann and Pfeifer, 2003; Hofacker et al., morphogen signal, BHL 2004). However, as in various organisms, the functionality of other proteins encoded by the gas vesicle genetic locus The 16.6 kb gas vesicle morphogenesis locus in S39006 of S39006 is unknown. is predicted to contain two operons, beginning with gvpA1 Here, we describe a comprehensive analysis of the and gvrA respectively. Reverse transcription polymerase 16.6 kb gas vesicle cluster in S39006. In frame deletions chain reaction (PCR) and 5′-RACE analysis showed that were created for each of the 19 genes in the cluster and the two operons did not overlap and that there was no impacts on gas vesicle production investigated. Pressure detectable read through between them (Fig. S2). The cor- nephelometry was used to determine the collapse pres- responding transcriptional start sites upstream of gvpA1 sure of gas vesicles, and volumetric occupancy of the and gvrA were determined (Fig. 2). Using reporter gene bacterial cytoplasm by vesicles in S39006, and an engi- fusions, we showed previously that transcription of gvpA1 neered E. coli-carrying gas vesicles, was assessed. We is controlled by the σ54, NtrC-like regulator GvrA. This is also exploited gas vesicles to calculate the instantaneous consistent with the location of a consensus σ54 binding turgor pressure of both S39006 cells and the recombinant site lying upstream of the gvpA1 transcriptional start site. E. coli strain by examining the collapse pressure of gas The production of gas vesicles in S39006 is cell density vesicles under different growth conditions. This study pre- dependent, controlled by the quorum-sensing locus, sents a complete analysis of a single gas vesicle gene SmaIR (Ramsay et al., 2011). SmaI produces the diffus- cluster and further demonstrates how gas vesicles can be ible signalling molecule, N-butanoyl-L-homoserine exploited in E. coli, generating cells that are composed of lactone (BHL) and, above a concentration threshold, BHL ∼50% gas vesicles. binds to SmaR, derepressing target gene expression (Slater et al., 2003). In a smaI mutant (BHL−), gas vesicles Results are absent but production can be restored by the addition of 1 μM BHL (Ramsay et al., 2011). BHL is therefore The core gas vesicle gene composition in defined as a diffusible morphogen. To determine whether proteobacteria SmaR directly regulated expression of gas vesicles S39006 carries a 16.6 kb cluster of 19 genes responsible through either the gvrA or gvpA1 operons, their respective for gas vesicle synthesis (Ramsay and Salmond, 2012). promoters were first inserted upstream of a This gas vesicle gene composition is similar to that β-galactosidase (β-gal) reporter fusion. We investigated of other proteobacteria, particularly β-, γ- and the expression of these two promoters in E. coli carrying δ-proteobacteria, possessing a gas vesicle gene cluster either the gvrA or gvpA1 promoter in the presence of a (Fig. 1). S39006 has three genes (gvpA1, gvpA2 and plasmid encoding SmaR, or with an empty vector control. gvpA3), encoding isoforms of the primary gas vesicle In the presence or in the absence of SmaR, β-gal expres- structural protein, GvpA; this is consistent with six sion from the gvrA promoter was the same (Fig. S2). In proteobacteria that possess a gas vesicle gene cluster. A contrast, activity of the gvpA1 promoter decreased by phylogenetic tree of GvpA showed that GvpA2 and GvpA3 over 75% in the presence of SmaR, but this repression are homologous to proteins previously annotated as could be relieved by the addition of 1 μM BHL (Fig. 2).
Load more

Recommended publications
  • The Methanosarcina Barkeri Genome
    Lawrence Berkeley National Laboratory Lawrence Berkeley National Laboratory Title The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes Permalink https://escholarship.org/uc/item/3g16p0m7 Authors Maeder, Dennis L. Anderson, Iain Brettin, Thomas S. et al. Publication Date 2006-05-19 Peer reviewed eScholarship.org Powered by the California Digital Library University of California LBNL-60247 Preprint Title: The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes Author(s): Dennis L. Maeder, Iain Anderson, et al Division: Genomics November 2006 Journal of Bacteriology Maeder et al. May 18, 2006 1 2 The Methanosarcina barkeri genome: comparative analysis 3 with Methanosarcina acetivorans and Methanosarcina mazei 4 reveals extensive rearrangement within methanosarcinal 5 genomes 6 7 8 9 Dennis L. Maeder*, Iain Anderson†, Thomas S. Brettin†, David C. Bruce†, Paul Gilna†, 10 Cliff S. Han†, Alla Lapidus†, William W. Metcalf‡, Elizabeth Saunders†, Roxanne 11 Tapia†, and Kevin R. Sowers*. 12 13 * University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 14 Columbus Center, Suite 236, 701 E. Pratt St., Baltimore, Maryland 21202, USA 15 † Microbial Genomics, DOE Joint Genome Institute, 2800 Mitchell Drive, B400, Walnut 16 Creek, CA 94598, USA 17 ‡ University of Illinois, Department of Microbiology, B103 Chemical and Life Sciences 18 Laboratory, 601 S. Goodwin Avenue, Urbana, Illinois 61801, USA 19 20 Running title: Comparative analysis of three methanosarcinal genomes 21 22 Keywords: Methanosarcina barkeri, archaeal genome, methanogenic Archaea 1 Maeder et al. May 18, 2006 23 ABSTRACT 24 25 We report here a comparative analysis of the genome sequence of 26 Methanosarcina barkeri with those of Methanosarcina acetivorans and 27 Methanosarcina mazei.
    [Show full text]
  • The Development of Bacterial Magnetic Resonance Imaging for Microbiota Analyses
    Western University Scholarship@Western Electronic Thesis and Dissertation Repository 9-11-2020 12:00 PM The development of bacterial magnetic resonance imaging for microbiota analyses Sarah C. Donnelly, The University of Western Ontario Supervisor: Burton, Jeremy P., The University of Western Ontario Co-Supervisor: Goldhawk, Donna E., The University of Western Ontario A thesis submitted in partial fulfillment of the equirr ements for the Master of Science degree in Microbiology and Immunology © Sarah C. Donnelly 2020 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Bacteria Commons, Medical Biophysics Commons, and the Medical Microbiology Commons Recommended Citation Donnelly, Sarah C., "The development of bacterial magnetic resonance imaging for microbiota analyses" (2020). Electronic Thesis and Dissertation Repository. 7381. https://ir.lib.uwo.ca/etd/7381 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Current microbial analyses to assess either the commensal microbiota or microorganism infection and disease typically require ex vivo techniques that risk contamination and are not undertaken in real time. The possibilities for employing imaging techniques in the microbiology field is becoming more prominent as studies expand on the use of positron emission tomography, ultrasound and numerous microscopy techniques. However, magnetic resonance imaging (MRI), a non-invasive in vivo modality that can produce real-time results is falling behind. Here, we examined the feasibility of detecting bacteria using clinical field strength MRI.
    [Show full text]
  • Phylogenomic Networks Reveal Limited Phylogenetic Range of Lateral Gene Transfer by Transduction
    The ISME Journal (2017) 11, 543–554 OPEN © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE Phylogenomic networks reveal limited phylogenetic range of lateral gene transfer by transduction Ovidiu Popa1, Giddy Landan and Tal Dagan Institute of General Microbiology, Christian-Albrechts University of Kiel, Kiel, Germany Bacteriophages are recognized DNA vectors and transduction is considered as a common mechanism of lateral gene transfer (LGT) during microbial evolution. Anecdotal events of phage- mediated gene transfer were studied extensively, however, a coherent evolutionary viewpoint of LGT by transduction, its extent and characteristics, is still lacking. Here we report a large-scale evolutionary reconstruction of transduction events in 3982 genomes. We inferred 17 158 recent transduction events linking donors, phages and recipients into a phylogenomic transduction network view. We find that LGT by transduction is mostly restricted to closely related donors and recipients. Furthermore, a substantial number of the transduction events (9%) are best described as gene duplications that are mediated by mobile DNA vectors. We propose to distinguish this type of paralogy by the term autology. A comparison of donor and recipient genomes revealed that genome similarity is a superior predictor of species connectivity in the network in comparison to common habitat. This indicates that genetic similarity, rather than ecological opportunity, is a driver of successful transduction during microbial evolution. A striking difference in the connectivity pattern of donors and recipients shows that while lysogenic interactions are highly species-specific, the host range for lytic phage infections can be much wider, serving to connect dense clusters of closely related species.
    [Show full text]
  • Demand in Vivo Monitoring of Tumor-Homing Bacteria Robert C
    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441537; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Genomically Mined Acoustic Reporter Genes Enable On- Demand In Vivo Monitoring of Tumor-Homing Bacteria Robert C. Hurt1,#, Marjorie T. Buss2,#, Katie Wong2, Daniel P. Sawyer1, Margaret B. Swift2, Przemysław Dutka1,2, David R. Mittelstein3, Zhiyang Jin3, Mohamad H. Abedi1, Ramya Deshpande2, Mikhail G. Shapiro2,* 1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA 3Division of Engineering and Applied Sciences, California Institute of Technology, Pasadena, CA, USA #Equal contribution *Correspondence should be addressed to M.G.S. ([email protected]) ABSTRACT A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is the difficulty of visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and a spatial resolution below 100 µm. Recently, the first genetically encoded reporter molecules were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging in the in vivo context. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics.
    [Show full text]
  • Acoustically Modulated Magnetic Resonance Imaging of Gas-Filled Protein Nanostructures
    SUPPLEMENTARY INFORMATIONARTICLES https://doi.org/10.1038/s41563-018-0023-7 In the format provided by the authors and unedited. Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures George J. Lu 1, Arash Farhadi 2, Jerzy O. Szablowski1, Audrey Lee-Gosselin1, Samuel R. Barnes 3, Anupama Lakshmanan2, Raymond W. Bourdeau 1 and Mikhail G. Shapiro 1* 1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA. 2Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA. 3Department of Radiology, Loma Linda University, Loma Linda, CA, USA. *e-mail: [email protected] NATURE MATERIALS | www.nature.com/naturematerials © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures George J. Lu1, Arash Farhadi2, Jerzy O. Szablowski1, Audrey Lee-Gosselin1, Samuel R. Barnes3, Anupama Lakshmanan2, Raymond W. Bourdeau1, Mikhail G. Shapiro1* 1Division of Chemistry and Chemical Engineering, 2Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. 3Department of Radiology, Loma Linda University, Loma Linda, CA. * Correspondence should be addressed to M.G.S. ([email protected]) Supplementary Information Theoretical consideration of the T2/T2* relaxation produced by gas vesicles Relaxation theory1-7 describes the T2/T2* relaxation of water near a contrast agent in three primary regimes: (1) the motional averaging regime, where ��# ∙ �& ≪ 1; (2) the static dephasing regime, where ��# ∙ �& ≫ 1; and (3) the intermediate regime, where ��# ∙ �& ~ 1. Here Δωr is the root-mean-square frequency shift at the surface of the contrast agent and τD is the time for a water molecule to diffuse across the distance of the contrast agent’s radius.
    [Show full text]
  • Microbial Gas Vesicles As Nanotechnology Tools: Exploiting Intracellular Organelles for Translational Utility in Biotechnology, Medicine and the Environment
    REVIEW Hill and Salmond, Microbiology 2020;166:501–509 DOI 10.1099/mic.0.000912 Microbial gas vesicles as nanotechnology tools: exploiting intracellular organelles for translational utility in biotechnology, medicine and the environment Amy M. Hill and George P. C. Salmond* Bacterium/archaeon Purify gas vesicles Apply ultrasound Display antigens MRI contrast Gas vesicle collapse Graphical abstract Gas vesicles are produced by a wide range of bacteria and archaea. Once purified they can be used to display antigens in vac- cines and as ultrasound contrast agents. Gas vesicle collapse is also a possible method to control cyanobacterial blooms. Abstract A range of bacteria and archaea produce gas vesicles as a means to facilitate flotation. These gas vesicles have been purified from a number of species and their applications in biotechnology and medicine are reviewed here. Halobacterium sp. NRC-1 gas vesicles have been engineered to display antigens from eukaryotic, bacterial and viral pathogens. The ability of these recom- binant nanoparticles to generate an immune response has been quantified both in vitro and in vivo. These gas vesicles, along with those purified from Anabaena flos- aquae and Bacillus megaterium, have been developed as an acoustic reporter system. This system utilizes the ability of gas vesicles to retain gas within a stable, rigid structure to produce contrast upon exposure to ultrasound. The susceptibility of gas vesicles to collapse when exposed to excess pressure has also been proposed as a bio- control mechanism to disperse cyanobacterial blooms, providing an environmental function for these structures. 000912 © 2020 The Authors This is an open- access article distributed under the terms of the Creative Commons Attribution License.
    [Show full text]
  • Direct Observation of Protein Secondary Structure in Gas Vesicles by Atomic Force Microscopy
    2432 Biophysical Journal Volume 70 May 1996 2432-2436 Direct Observation of Protein Secondary Structure in Gas Vesicles by Atomic Force Microscopy T. J. McMaster,* M. J. Miles,* and A. E. Walsbyt *H. H. Wills Physics Laboratory, and *Department of Botany, University of Bristol, Bristol, BS8 1TL England ABSTRACT The protein that forms the gas vesicle in the cyanobacterium Anabaena flos-aquae has been imaged by atomic force microscopy (AFM) under liquid at room temperature. The protein constitutes "ribs" which, stacked together, form the hollow cylindrical tube and conical end caps of the gas vesicle. By operating the microscope in deflection mode, it has been possible to achieve sub-nanometer resolution of the rib structure. The lateral spacing of the ribs was found to be 4.6 ± 0.1 nm. At higher resolution the ribs are observed to consist of pairs of lines at an angle of -55° to the rib axis, with a repeat distance between each line of 0.57 ± 0.05 nm along the rib axis. These observed dimensions and periodicities are consistent with those determined from previous x-ray diffraction studies, indicating that the protein is arranged in ,B-chains crossing the rib at an angle of 550 to the rib axis. The AFM results confirm the x-ray data and represent the first direct images of a ,3-sheet protein secondary structure using this technique. The orientation of the GvpA protein component of the structure and the extent of this protein across the ribs have been established for the first time. INTRODUCTION Gas vesicle protein and structure Gas vesicles are hollow structures that provide buoyancy in volume of this unit cell, 10.25 nm3, corresponds with that of various aquatic microorganisms.
    [Show full text]
  • Study of the Archaeal Motility System of H. Salinarum by Cryo-Electron Tomography
    Technische Universität München Max Planck-Institut für Biochemie Abteilung für Molekulare Strukturbiologie “Study of the archaeal motility system of Halobacterium salinarum by cryo-electron tomography” Daniel Bollschweiler Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. B. Reif Prüfer der Dissertation: 1. Hon.-Prof. Dr. W. Baumeister 2. Univ.-Prof. Dr. S. Weinkauf Die Dissertation wurde am 05.11.2015 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 08.12.2015 angenommen. “REM AD TRIARIOS REDISSE” - Roman proverb - Table of contents 1. Summary.......................................................................................................................................... 1 2. Introduction ..................................................................................................................................... 3 2.1. Halobacterium salinarum: An archaeal model organism ............................................................ 3 2.1.1. Archaeal flagella ...................................................................................................................... 5 2.1.2. Gas vesicles .............................................................................................................................. 8 2.2. The challenges of high salt media and low dose tolerance in TEM .........................................
    [Show full text]
  • Cellular Solid-State Nuclear Magnetic Resonance Spectroscopy
    Cellular solid-state nuclear magnetic SEE COMMENTARY resonance spectroscopy Marie Renaulta, Ria Tommassen-van Boxtelb, Martine P. Bosb, Jan Andries Postc, Jan Tommassenb,1, and Marc Baldusa,1 aBijvoet Center for Biomolecular Research, bDepartment of Molecular Microbiology, Institute of Biomembranes, and cDepartment of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Edited by Robert Tycko, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board January 6, 2012 (received for review October 11, 2011) Decrypting the structure, function, and molecular interactions of conditions (11) as a high-resolution method to investigate atomic complex molecular machines in their cellular context and at atomic structures of major cell-associated (macro)molecules. resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well- Results established imaging method that can visualize cellular entities at Sample Design for Cellular ssNMR Spectroscopy. Our goal was to the micrometer scale and can be used to obtain 3D atomic struc- establish general expression and purification procedures that lead 13 15 tures under in vitro conditions. Here, we introduce a solid-state to uniformly C, N-labeled preparations of whole cells (WC) NMR approach that provides atomic level insights into cell-asso- and cell envelopes (CE) containing an arbitrary (membrane) ciated molecular components. By combining dedicated protein pro- protein target (Fig. 1B). As our model system, we selected the duction and labeling schemes with tailored solid-state NMR pulse 150-residue integral membrane-protein PagL from Pseudomonas methods, we obtained structural information of a recombinant aeruginosa, an OM enzyme that removes a fatty acyl chain from integral membrane protein and the major endogenous molecular LPS (12).
    [Show full text]
  • The Transcriptional Activator Gvpe for the Halobacterial Gas Vesicle
    Article No. mb981795 J. Mol. Biol. (1998) 279, 761±771 The Transcriptional Activator GvpE for the Halobacterial Gas Vesicle Genes Resembles a Basic Region Leucine-zipper Regulatory Protein Kerstin KruÈ ger1, Thomas Hermann2, Vanessa Armbruster1 and Felicitas Pfeifer1* 1Institut fuÈr Mikrobiologie und The GvpE protein involved in the regulation of gas vesicles synthesis in Genetik, Schnittspahnstr. 10 halophilic archaea has been identi®ed as the transcriptional activator for Technische UniversitaÈt the promoter located upstream of the gvpA gene encoding the major gas Darmstadt, D-64287 vesicle structural protein GvpA. A closer inspection of the GvpE protein Darmstadt, Germany sequence revealed that GvpE resembles basic leucine-zipper proteins typically involved in the gene regulation of eukarya. A molecular model- 2Max-Planck-Institut fuÈr ling study of the C-terminal part implied a cluster of basic amino acid Biochemie, D-82152 residues constituting the DNA-binding site (DNAB) followed by an Martinsried, Germany amphiphilic helix, suitable for the formation of a leucine-zipper structure within a GvpE dimer. The model of a GvpE dimer docked onto DNA indicated that the side-chains of the basic residues could perfectly interact with the negatively charged phosphate groups of the DNA backbone. Substitution of three basic amino acid residues of this putative DNAB by alanine and/or glutamate generated mutated GvpE proteins. None of these was able to activate the c-gvpA promoter in vivo, indicating that these basic residues are required for GvpE activity. This identi®cation of an archaeal gene regulator displaying similarity to eukaryal regulatory proteins implies that the basic transcription machinery of eukarya and archaea are closely related, and that the regulatory proteins have evolved according to common principles.
    [Show full text]
  • Whole‐Genome Comparison Between the Type Strain Of
    Received: 17 July 2019 | Revised: 8 November 2019 | Accepted: 9 November 2019 DOI: 10.1002/mbo3.974 ORIGINAL ARTICLE Whole-genome comparison between the type strain of Halobacterium salinarum (DSM 3754T) and the laboratory strains R1 and NRC-1 Friedhelm Pfeiffer1 | Gerald Losensky2 | Anita Marchfelder3 | Bianca Habermann1,4 | Mike Dyall-Smith1,5 1Computational Biology Group, Max-Planck- Institute of Biochemistry, Martinsried, Abstract Germany Halobacterium salinarum is an extremely halophilic archaeon that is widely distributed 2 Microbiology and Archaea, Department of in hypersaline environments and was originally isolated as a spoilage organism of Biology, Technische Universität Darmstadt, T Darmstadt, Germany salted fish and hides. The type strain 91-R6 (DSM 3754 ) has seldom been studied 3Biology II, Ulm University, Ulm, Germany and its genome sequence has only recently been determined by our group. The exact 4 CNRS, IBDM UMR 7288, Aix Marseille relationship between the type strain and two widely used model strains, NRC-1 and Université, Marseille, France R1, has not been described before. The genome of Hbt. salinarum strain 91-R6 consists 5Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, of a chromosome (2.17 Mb) and two large plasmids (148 and 102 kb, with 39,230 bp University of Melbourne, Parkville, Vic., being duplicated). Cytosine residues are methylated (m4C) within CTAG motifs. The Australia genomes of type and laboratory strains are closely related, their chromosomes shar- Correspondence ing average nucleotide identity (ANIb) values of 98% and in silico DNA–DNA hy- Friedhelm Pfeiffer, Computational Biology Group, Max-Planck-Institute of bridization (DDH) values of 95%. The chromosomes are completely colinear, do not Biochemistry, Martinsried, Germany.
    [Show full text]
  • Reporter Genes for Ultrasound and Magnetic Resonance Imaging
    Available online at www.sciencedirect.com ScienceDirect Proteins, air and water: reporter genes for ultrasound and magnetic resonance imaging 1,4 2,4 3 George J Lu , Arash Farhadi , Arnab Mukherjee and 1 Mikhail G Shapiro A long-standing goal of molecular imaging is to visualize this limitation and predicted that ‘the prevalence and cellular function within the context of living animals, success of GFP indicate that comparable revolutions necessitating the development of reporter genes compatible might result from genetic sequences that robustly encode with deeply penetrant imaging modalities such as ultrasound image contrast for other methods’ [1]. Indeed, reporter and magnetic resonance imaging (MRI). Until recently, no genes for noninvasive deep-tissue imaging modalities reporter genes for ultrasound were available, and most such as ultrasound and magnetic resonance imaging genetically encoded reporters for MRI were limited by metal (MRI) could have great value in both basic biomedical availability or relatively low sensitivity. Here we review how research and the development of cellular diagnostics and these limitations are being addressed by recently introduced therapeutics. However, no such reporter genes have so far reporter genes based on air-filled and water-transporting achieved the prevalence of GFP, and new ideas are biomolecules. We focus on gas-filled protein nanostructures therefore needed to spark the envisioned revolutions. adapted from buoyant microbes, which scatter sound waves, perturb magnetic fields and interact with hyperpolarized nuclei, This review summarizes progress on two recently intro- as well as transmembrane water channels that alter the duced classes of genetically encoded contrast agents for effective diffusivity of water in tissue.
    [Show full text]